PROJECT SUMMARY T2R bitter taste receptors are G-protein coupled receptors originally identified on the tongue. Human nasal and bronchial airways express multiple T2Rs isoforms (T2R4, T2R14, T2R16, and T2R38) within motile cilia. These T2Rs recognize bacterial products and, when activated, stimulate a signaling cascade involving calcium-driven nitric oxide production that increases ciliary beating as well as directly kills bacteria. We hypothesize that common genetic polymorphisms in TAS2R genes altering receptor function may underlie susceptibility to infection, a notion supported by recent clinical data correlating nonfunctional TAS2R38 with susceptibility to chronic rhinosinusitis (CRS). We also hypothesize that activation of T2R bitter receptor responses in airway ciliated cells (and possibly other epithelia where they are expressed) will activate innate immune to eradicate infections without the use of conventional antibiotics, avoiding pressures for resistance. Development of T2R agonists as topical therapeutics for respiratory infections will be facilitated by further knowledge of the signaling pathways and downstream effects of these receptors. Some bitter molecules have also been reported to be anti-inflammatory in several previous studies, but this has not been linked to T2R activation. We hypothesized that cross-talk between signaling may exist between T2Rs and toll- like receptors (TLR), a major class of pattern recognition receptors (PRRs) expressed in airway cells that frequently signal through the transcription factor NF?B to activate inflammation. Our preliminary data suggest that T2R activation has suppressive effects on TLR-mediated cytokine secretion in airway epithelial cells. Likewise, and surprisingly, TLR stimulation had acute suppressive effects on T2R-activated calcium and nitric oxide. The overall aim of this grant is to elucidate the mechanisms that underlie these effects. The discovery of cross-talk between these two signaling pathways has wide-spread implications for many tissues due to the wide expression of T2Rs and TLRs. A combination of bactericidal innate immune responses and anti- inflammatory effects may make T2R agonists beneficial to patients with inflammatory airway diseases. In Aim 1, we will examine the effects of T2Rs on TLR-mediated NF?B activation and cytokine secretion. In Aim 2, we will examine the acute effects of TLR stimulation on T2R responses. We will use primary human nasal cells isolated from residual surgical material and grown and differentiated at air-liquid interface combined with live cell imaging, biochemistry, and molecular biology. This project will establish a necessary baseline level of understanding regarding molecular mechanisms of cross-talk between these immune signaling pathways necessary to develop future projects aimed at understanding how this cross talk fits within the context of airway infection and inflammation. This project will also reveal fundamental insights into airway host-pathogen interactions.